Note: Descriptions are shown in the official language in which they were submitted.
SPECIFICATION 2~3~LfL~70
BACKGROUND OF THE lNVENTION
Field of the Invention
This invention relates to an immunoassay of elas~ase-l which is a
proteolytic en~yme secreted from the panc:reas and contained in body
fluids. This immunoassay is useful for t:he diagnosis of p~ncre~tic
diseases. Further, this invention relates to the human elastflse-l
treated with a compound of the formula:
(CH~) 3COCO[ NHCH(CH~)CO]3CH2Cl
(hereinafter abbreviated as Boc-TACK), which is useful for said
immunoassay.
Prior Art
The incidence of pancreatic diseases hss been sho~ing an
increasing tendency in recent years due to various reasons and this
type of diseases is now serious in a clinical fi ld. However, since
the pancreas is situated in the deepest part of the abdominal cavity,
it is difficult to detect said diseases and there has been 8 d~m~nd
for the development of a simple and accurate method of diagnosis
therefor.
As biochemical methods of diagnosis for pancreatic diseases, a
method is kno~n in which amylase in the patient's body fluids (mainly
blood and urine, and also pancreatic Juice, pleural dropsy and
abdominal dropsy) is measured. However, the blood amylase measured by
this method shows the total amount of two amylases, one originating
from the salivary gland and the other from the pancreas Therefore,
this method of measuring amylase is no~ regarded as a specific
diagnostic method or pancreatic diseases. Besides, the period in
which amylase show; high values after onse~ of the pancreatic diseases
is short, and when this period is over, it is impossible to make ~he
~ 3L~ 3
diagnosis. For this reason, there has been a demand for the
development of some other method for diagnosis of pancreatic diseases
by messuring pancreatic enzymes.
Human pancreas or pancrestic juice contsins, in addition to
amylase, elastase which is an enzyme decomposing elastin, a fibrous
protein. There are two types of elastase. One is elastase-l
(molecular weight 29,000 - 33,000) with acid charge, and the other is
elastase-~ (molecular weight 25,000 - 26,000) with basic charge.
Elastase-l activates elas~ase-2. These elastases are diferent from
each other in the aspects of enzymology, protein chemistry and
immunology. The measurement of this elastase-l is useful in the
diagnosis and observ~tion of subsequent development of the p~ncreatic
diseases, especially acute pancreatitis, chronic and recurre.nt
pancreatitis, and cancer of the pancreas. Serum elastase-l is known
to increase remarkably in acute pancreati~is. It is also known to
increase more remarkably than other pancreatic enzymes in cQncer of
the pancreas.
Attempts have hitherto been made to measure elastase-l based upon
its enzymatic activity. However, it has been difficult to measure
elastase-l by this method because of the following reasons: The amount
of elastase contained in blood is very small. In addition, the blood
contains a large amount of elastase inhibitors, a l-antitrypsin and
a 2-macroglobulin, and these inhibit elastase from showing its
activity.
As sho~n above, elastase is present in blood, being bound to
inhibitors, a ,-antitrypsin and a 2-macroglobulin. a l-ankitrypsin is
bound to the active site of elastase, but the structure itself of
elastase remains unchanged. Therefore, it has an immunological
activity, and ca~ take part in antigen-antibody reaction. On the
other hand, the iDhibition by a 2-macroglobulin is done in a form of
~0~4~7~3
masking elastase. As a result, it becomes impossible for antibodies
to recognize the structure of elastase and, consequently, it becomes
impossible to measure elastase even by immunological procedures.
Therefore, attempts were made to measure, by an immunological method,
the elastase bound to ~ I-antitrypsin in the blood. In immunoassay,
however, the immunological activity of the labeled elastase varies
depending on the amount of elastase inhibitor con~ained in the serum
specimen. Consequently, it was difficult to make accurate qusntitative
determination of elastase.
Meanwhile, it was found that, when the labeled elastase was
pretreated with diisopropyl fluorophosphate (DFP), a synthetic
inhibitor of elastase, it became impossible for the elastRse inhibitor
in the blood to bind to the labeled elastase. Consequently, a method
for the immunoassay of elsstase-l with the use of the aforesaid DFP-
treated elastase-1 was developed (Japanese Patent P~blic~ion No.
25183/1984). There was also developed a method (Japanese Patent
Unexamined Publication No. 73152/1988) in which labeled elastase-1
treated with DFP, phenylmethanesulfonyl fluoride (PMSF), p-
chloromercuribenzoate (PCMB) or a l-antitrypsin and anti-elastase
monoclonal antibody are used.
As synthetic inhibitors which inhibit the elastase from being
bound to the inhibitors in the blood while maintaining the
immunological activity of the elastase, there are known DFP, PMSF and
PCMB. On the other hand, Boc-TACK is known to inhibit the enzymatic
activity of porcine elastase (FEBS LETIERS Vol. 67, 156-1~0 (1976)).
However, it has not been known that Boc-TACK inhibits the enzymatic
activity of human elastase. Nor has it been known that Boc-TACK
inhibits the elast~se-l from being bound to a ,-antitrypsin~
In the conventional method of immunoassay, elastase inhibitors
such as a ,-antitrypsin and a 2-macroglobulin in the serum bind to
2(~LfL'7(3-'3
labeled elastase-l, thereby inhibiting the immNnological acti~ity of
the labeled elastase-l. Therefore, it was impossible to make ~ccurate
quantitative determination of blood elastase-l.
For the prevention of the binding of a ,-antitrypsin snd a 2-
macroglobulin to elastase-l, a method is known in which the active
group of labeled elastsse-l is blocked by DFP, PMSF and PC~B
(mentioned above). However, of these synthetic inhibitors, PMSF is
weak in capability of inhibiting the binding of a ,-antitrypsin and
2-macroglobulin to elastase-l.
Besides, DFP and PMSF are toxic in tha~ they contsin fluorine and
iherefore generate hydrogen fluoride when binding to elastase-l.
Furthermore, DFP causes disturbance of the optic nerve. Therefore,
care must be taken when using these materials.
Meanwhile, PCMB contains mercury, so its use may bring about
environmental pollution.
For these reasons, there has been a demand for a safe synthetic
inhibitor free from toxic substances such as fluorine and mercury
which completely inhibits the binding of inhibitors in the blood to
elastase-l without disturbing the im~unological activity of the
elastase-l and which can take the place of PMSF and DFP.
SUMMARY
This invention provides an immunoassay of elastase-l, ~hich
comprises subjecting (a) labeled elastase-l treated with Boc-TACK and
(b) elastase 1 to be measured to competitive reaction ~ith anti-
elastase-l antibody, wherein the Boc-TACK denotes
(CH3)3C9COENHCH(CH3)CO]~CH2Cl.
The synthetic inhibitor Boc-TA~K almost completely inhibits the
binding of a ,-antitrypsin and a 2-m~croglobulin to elastase-l without
disturbing the i~munological activity of the elastase-l and realizes
_~_
~o~L~7a33
a simple and accurate immunoassay of elastase-l. Further, since the
Boc-TACK generates no toxic hydrogen fluoride in the ~reatment of
elastase-l therewith and contains no harmful mercury, it is much safer
to use than conventional inhibitors such as PCMB, PMSF and DFP.
This invention further provides the Boc-TACK-treated human
elastase-l, which essential~y does not react with a 1- antitrypsin or
with a 2 -macroglobulin.
BRIEF DESCRIPTION OF THE DRAWI~GS
Fig. 1 shows the results of the elution of '26I-labeled El(a) not
treated with Boc-TACK snd Boc-TACK-treated '2~I-labeled El(b), both in
human serum, from Ultrogel AcA 44 (1.5 x 55 cm) wi~h the use of 50 mM
phosphate buffer (pH 7.1).
Fig. 2 shows the effect of a l-antitrypsin and a 2-macroglobulin
on the rate of the binding of anti-El serum wi~h l25I El not treated
or treated with PMSF or Boc-TACK.
Fig. 3 gives standard curve of El and dilution curve of human
serum.
Fig. 4 illustrates the effect of Boc-TACK-treated El on standard
curve.
Fig. 5 shows cross reactivity of El and El complexes containing
a l antitrypsin or a 2-ma~roglobulin.
Fig. 6 shows cross reactivity with several animsl sera.
DE~AlLED DE~CRIPTION OF THE INYENTION
In order to search for a synthetic inhibitor which is superior to
PMSF and DFP and which almost completely inhibits the binding of a 1-
antitrypsin and a ~-macroglobulin to elastase-l witho1~t disturbing the
immunological activity of the elastase-l so as to establish a better
and more accurate method for the measurement of elastase-l, the
-5
Z O~L~ 3
inventors made strenuous efforts and studies and, as a result~ have
completed a method for the immunoassay of elastase-l charscterized by
the use of Boc-TACK, a synthetic inhibitor.
The term Boc-TACK as used in this speciication is an
abbreviation (Boc lri-Alanyl Chloromethy:L Ketone) of N a - [ N-(tert-
butoxycarbonyl)alanyl]-N-(3-chloro-1-methyl-2-oxopro w l)alanine~mide
having the following formula:
(CH3)~COCO[NHCH(CH~)CO]~CH2Cl
wherein the alanine residues are prefera~ly of B configur~tion.
The human elsstase-l (EC 3.4.21.11) used in this invention may be
obtained by purifying elastase-l obtained from the human pancreatic
juice in accordance with conventional methods such as the method of
Feinstein et al. (Eur. J. Biochem. 43, 569 (1974)). A lsbel for the
humQn elastase-l obtained may be chosen from among conventional
labeling agents such as radioisotope, enzyme, fluorescent substance
and luminescent substance. For labeling by radioactive iodine,
chloramine T method, lactoperoxidase method or Bolton-Hunter method
may be employed. As for radioactive iodine, '2sI and 19~I are used.
As labeling agents, there are used R -galactosidase, horseradish
peroxidase, glucose oxidase, slksline phosph~tase, glucose-6-
phosphatase, dehydrogenase, etc. As fluorescent substances, there are
used substances of fluorescein type such as fluor2scein iso~hiocyanate
and materials of rhodamine type such as tetramethylrhodamine
isothiocyanate. In this invention, it is especially preferable to
label with '2~I by the chloramine T method.
The labeled human elastase-l obtained is processed into
Boc-TACK-bound labeled human elastase-l by the treatment with
Boc-TACK. This process may be performed, for example, by adding
labeled human elastase-l into a suitable buffer solution, such ~s
phosphate buffer, containing Boc-TACK and then by allowing it to stand
--6--
21~Lf~0 3
overnight so as to cause Boc-TACK to be bound to the labeled human
elastase-l. Since the binding of Boc TACK to elastase-l does not
produce any hydrogen fluoride, this i5 a safe process.
As anti-elastase-l antibody, antisera may be obtained from mammal
such as rabbits, goats, chickens and guinea pigs i~muni~ed with human
elastase-l together with a suitable adjuvant such 8S Freund's complete
adjuvant. As anti-elastase-l antibody, a monoclonal antibody m~y also
be used.
The immunoassay of elastase-l of this invention is characterized
in that the labeled human elastase-l treated with Boc-TACK as above
and the elastase-l to be measured, human elastase-l for example, are
subjecked to competitive reaction with above-mentioned anti-elastase-l
antibody.
After the competitive r~action, the labeled human el~stase-l to
which anki-elastase-l antibody had been bound and the free labeled
human elastase-l are separated by a conventional method ~-o obtain each
fraction thereof, and then the activity of the labeling agent in
either or both of said fractions is measured. ~eneraliy, it is enough
to ~easure the activity in ~he antibody-bound fraction.
The aforesaid separation of the frac~ions may be performed by
known methods such as solid phase method, sandwich method and double
antibody method. However, in Examples of this invention, the double
antibody method was us~d for the separation. As for second antibody
used in the double antibody method, immuno-besds-goat anti-rabbit IgG
antibody may be used where rabbit serum i5 used as an anti-el~stase-l
antibody.
~ he values obtained from the measure~ent of the lPbeling agent in
the above fractions are compared with the standsrd curve obtained with
the standard elastase-l having known concentrations, whereby the
amount of the elastase-l to be measured is calculated.
Z 03L~L'7C3
This invention further relates to the Boc-TACK-treated human
elastase-l obtained as above. As shown in Examples given herein
later, the Boc-TACK-treated human elastase-l of this invention
essentially does not react with a l-antitrypsin or with a 2-
macroglobulin. Besides, no toxic hydrogen fluoride is generated in
treatment with Boc-TACK. Nor does Boc-TACX contain any harmful
mercury.
Furthermore, DFP and PMSF bind to all serine resid~es, whereas
Boc-TACK binds specifically to the active center of elastase-l. Thus,
the immunological activity of elastase-l is little affected by
Boc-TACK.
Therefore, Boc-TACK is suitable for use in the meas~rement system
where a ,-antitrypsin or a 2-macroglobulin is present. It becomes
even more useful when it is labeled beforehand with an enzyme,
radioisotope or fluorescent subst~nce.
2~)3l4~(3~3
Example 1
Synthesis of elastase inhibitor Boc-TACK:
(CH~)~COCO[NHCH(CH3)CO]3CH2Cl (I)
_
The above-mentioned tripeptide derivstive was prepared according
to the following procedures. Hereinafter, Boc-TACK is referred to as
Boc-Ala-Ala-Ala-CH2Cl.
H-Als-OBzl
~ Boc-Ala-OSu
Boc-Ala-Ala-OBzl (II)
2O%-TFA/CH2Cl2
~ Boc-Ala-OSu, DIEA
Boc-Ala-Ala-Ala-OBzl (III)
~ H2/Pd
Boc-Ala-Ala-Ala-OH (IV)
ClCOe CH2 CH(CH3 ) 2 ~ N-methyl morpholine
~ CH2N2/(C2Hs)20
Boc-Ala-Ala-Ala-CH~ (V)
~ lM HCl/AcOH
H-Ala-Ala Ala-CH2Cl (VI)
~ (Bocj 20/DMF
Boc-Ala-Ala-Ala-CN2Cl (I)
Boc-Ala-Ala-OB~l (II)
In 10 ml of DMF (N,N-dimethylformamide) is dissolved H-Ala-OBzl
p-toluenesulfonate (2.0 g, 5.7 ~mol), which is then cooled wi~h ice.
To this are added equimolar M EA (N,N-diisopropylethylamine) and Boc-
Ala-OSu IHOSu: N-hydroxysuccinimide) (1.96 g, 6.8 mmol). The mixture
is allowed to undergo reactio~ for 20 hours at 25C, ater which the
solvent is evaporated under reduced pressure. The residue is
dissolved in ethyl acetate, which is then washed with 1 M HCl, 1 M
Z(~L~7(~
NaHCO~ and H20 in the order mentioned. After that, the ethyl acetate
layer is dried with MgSO,. The solvent is then evaporated, and the
crystals obtained are purified on a silica gel column (Kieselgel H,
50 g, CHCla: MeOH = 99 : l), followed by recrystalliza~ion from
ether-petroleum ether, whereby 1.85 g (~.3.0%) of compount II is
obtained.
m.p. 65 - 67~
[a ]D' - 26.9 + 0.7 ~c = 1.0, DMF)
Boc-Ala-Ala-Ala-OBzl (III)
-
To the compound II (1.8 g, 5.1 m~ol) obtained above is ~dded 20%
TFA (trifluoroacetic acid)/CH2Cl2, which i5 the~ allowed to undergo
reaction for 30 minutes st 25C. The solvent is evaporated, and the
residue is dissolved in 20 ml of DMF. The solution is cooled with
ice, which is then neutralized with DIEA. To this is added Boc-Ala-
OSu (1.65 g, 6.2 mmol), and the mixture is allowed to undergo reaction
for 20 hours at 25C. The reaction solution is treated in the same
manner as in the above-mentioned compound II. The crude product
obtained is purified with silica gel (Kieselgel H, 50 g, CHclJ: MeOH
= 98 : 2), followed by recrystalli~ation from ether to give 2.0 g
(93%) of compound III, m.p. 140 - 141C.
[a]D~ - 30.4 l 0.7 (c = 1.0, DMF)
Boc Ala-Als-Ala OH (IV)
Compound III (2.0 g, 4.75 mmol) obtained as above is subjected,
by a conventional method, to catalytic reduction in methanol for 5
hours with pallatium black (Pd black) as catalyst (H2 consu~ption ca.
103 ml). The solvent is evaporated and the residue obtained is
--1 0-
~ O~L~7 0 3
recrystallized from methanol-ether to give 1.56 g (9Ç~X) of the
compound IV, m.p. 200 - 201C (decomp.).
[ a]D' - 20.5 ~ 0.6~ (c = 1.0, DMF)
Boc-Ala-Ala-Ala-CHN2 (V)
Compound IV (1.03 g, 3.3 mmol) obtained as above is dissolved in
20 ml of anhydrous ~HF (tetrahydrofuran)~ which is then cooled to -
10C. To this are added 0.36 ml (3.3 mmol) of N-methylmorpholine and
then 0.45 ml (3.3 mmol) of isobutyl chloroformate ~nd the mixture is
stirred for 30 minutes at the same temperature. To this is added a
solution of di~zomethane in ether and the resction is allowed ~o t~ke
place for 30 minutes. After confirming that the yellow color o
diazomethane is still present, the solvent is evaporated under reduced
pressure. To the residue is added ether and the resulting gelatinous
precipitates are collected by filtration. The product obtained is then
purified with silica gel column (Kieselgel H, 50 g, CHMJ: MeOH = 98 :
2), followed by recryst~llization from ether, ~hereby 0.60 g (51YD) of
the compound V is obtained 8S yellow needles, m.p. 165C ~deco~p.~.
IR: 2108 (-CHN2), 1676 (~C = O) cm-
~'H NMR: 6.03 ppm ~-CO-CHN2)
.
H-Ala-Ala-Ala-CH2Cl. HCl (VT)
.__ _ _
Compound V (500 mg, 1.4 mmol) obtained as above is dissolved in 5
ml of 1 M HCl/AcON. The solution is kept at ODC for 30 minutes, and
then at 25C for 60 minutes, followed by evaporation to dryness under
reduced pressure. The residue is subjected to partition
chromatography on a Sephadex LH-20 column (4 x 35 cm), with 1-BuOH -
AcGH - EtOH - H20 (2:2:1:1) as solvent. Fractions containing the
desired product are collec~ed and the solvent is e~aporated to give
--1 1--
~ 01~703
200 mg of crude product. The thin-layer chromatography (Kieselgel Ht
AcOEt -AcOH - H20 = 4:1:1, ninhydrin coloration) of the crude product
shows two spots.
Boc-Ala-Ala-Ala-CH2Cl (I)
Compound Vl (ca. 100 mg) obtained as above is dissolved in 2 ml
of DMF, which is then neutralized ~ith M EA. To this is added (Boc)20
(100 mg, ca. 0.5 mmol) and the mixture i.s allowed to undergo re~ction
for 30 minutes at 25~C. Then, the solverlt is evaporated, and ~he
residue is purified on a silica gel column (Kieselgel H, 20 g, CHCl, :
MeOH = 98:2). Fractions containing the diseired product are
collected and the solvent is evaporated, followed by crgstallization
from ether to give 51 mg of the objective compound I, m.p. 20~'C
(decomp.).
[ a 1 D~7 - 90.8 + 2.6 (c = 0.5, MeOH~
1H NMR: 4.35 ppm (-C0-CH2Cl)
xample 2
Radioimmunoassay of human serum elastase-l with_ _ _
the use of Boc-TACK-bound '251- labeled elastase-l
. _ _
In case psncreatic elastase-l in blood (herein~fter abbreviated
as El) is measured by radioimmunoassay (compe~itive method), labeled
antigen ('26I-labeled El) binds to inhibitors, a ,-antitrypsin and
a 2-macroglobulin in the blood, whereby the antigen-antibody reaction
is inhibited. As a result, i~ becomes impossible to accurately measur
serum El. To prevent this, a method is known in which the active group
(binding site) of '26I-labeled El is blocked ~ith PMSF, DFP, etc. (as
mentioned above).
In the following Examples, '2~1-labeled El ~reated with Boc-TACK
~O~L~7 O 3
does not bind to the serum inhibitors and all of it can tQke part in
an~igen-antibody reaction. This was confirmed by gel filtration
method and also by immunological procedures. Thus, Boc-TACK proves
to be useful for the radioimmunoassay of human serum El.
I. Reagents and method
(l) Preparation of elastase
To 4Q of human pancreatic juice is added 2.8 kg (70% saturation)
of ammonium sulfate, which is then allowed to stand ~t 4C for 1 - 2
days. The precipitate is collected by centrifugation, which is
suspended in 200 ml of buffer solution 1 (50 mM sodium acetate and 10
mM CaCl2, pH 6.5), and then dialyzed for 2 days against ~he same
buffer solution (outer solution 5Q , replaced 4 times). To lower the
conductivity of the dialysis inner solution (775 ml), buffer solution
1 is added to bring the volume finally to 2.0Q (0.~5 mho, pH 6.45).
The mixture is charged to SP-Sephadex C-25 (2.5 x 41.0 cm) buffered
beforehand with buffer solution 1. It is then washed with buffer
solution 1 equivalent to 10 times the column volume, After which it is
eluted with buffer solution 1 equivalent to 15 times the vol~me of the
resin with a linear concentration gradient of 0 1 0.4 M. NaCl. In the
pass-through fraction were seen an activity of decomposing elastin and
an activity of decomposing succinyl-tri-alanina-p-hitroanilide
(hereinafter abbreviated as SA9PNA), both brought about by elastase.
However, in this fraction there were also found some trypsin and
chymotrypsin as well as elastase. Therefore, this fraction is
collected ~1295 ml)7 and to this is added 903 g (70% saturation) of
ammonium sulfate. ~he mixture is allowed to stand at 4~ for 3 days.
After that, the precipitate is collected, which is then dissolved in
about 40 ml of buffer solution l. This elastase fra~tion is dialyzed
for 4 days against buffer solu~ion 1 (outer solution 5Q , replaced
twice). Af~er bringing its volume to 82 ml, it is charged to Sepharose
~ L~7 0 3
4B-F Aminocaproyl-(Ala)3 (1.5 x 24.0 cm). The trypsin and chymotrypsin
mixed therein passed through completely, but elastase showed a
tendency of being adsorbed slightly. However, elastase fraction still
contains a small quantity of trypsin and chymotrypsin. ~o remove
these, elastase fractions are collected (147.0 ml), which is then
concentrated by ultrafiltration (Amicon, YM-10) to a volume of 45 ml.
It is then dialyzed for two days (outer solution 5Q , replac~d twice)
against buffer solution 2 (10 mM Tris-HCl and 2 mM C~Cl~, pH 7.5). The
volume finally comes to 57 ml, which is charged to Sepharose 4B-E
Aminocaproyl (Ala)3 (1.5 X 24.0 cm) buffered beforehand with bufer
solution 2. Elastase showed a tendency of being adsorbed slightly.
However, most of it was eluted in the vicinity of the passing fraction
. Chymotrypsin came out before elastase and trypsin after elastAse.
Thus, the trypsin and chymotrypsin mixed in elastase were removed
almost completely. There was obtained finally 52.8 mg of elast~se.
(2) Properties of elastase
(A) Purity
The uniformity of the obtained elastase was confirmed by each of
SDS-PAGE (15% acrylamide, 1 mm gel), pH 9.4 gel electrophoresis (7.5%
acrylamide, 1 mm gel), and pH 4.0 gel electrophoresis (7.5%
acrylamide, 1 mm gel).
(B) Molecular weight
When calculated from the transfer rate in the aforesaid SDS-PAGE
wi~h the use of electrophoresis calibration kit made by Pharmacia, the
molecular weight came to 315~0. This was larger than the ~olecular
weight of 25590 (Japanese Patent Unexamined Publicatio~ No.
111688/19~6) estimated of elastase-l based on cDNA. Since its amino
acid composition is nearly the same as that of the elastase-l
described in Jap~nese Paten~ Unexamined Publication No. 1116B8/1986,
it is presumed that the accurate molecular weight was not reflected in
-14-
x~
the aforesaid measurement of molecular weight by SDS-PAGE due to the
peculiarity of elastase molecule.
(C) Analysis of amino ~cids
The obtained elastase was desalted with HPLC (Brownlee, Aquapore
RP-300 column). The resulting product was hydrolyzed for 24 hours at
llO~C with the usa of 4 M methanesulfonic acid containing 0.2% 3-(2-
aminoethyl)indole. The analysis of ~mino acids ~as performed with
Hitachi Amino Acids Analyzer (Model 835), the results of which are
shown in Table 1. The amino acid composition obtained in the present
measurement W8S nesrly the same as that of elastase-l estim~ted from
cDNA.
~ 4~C~3
Table 1
Amino acids Results Reference*'
Asp ~2.0 21
Thr 12.8 13
Ser 19.8 19
Glu 19.9 19
Pro 15.9 16
Gly 24.8 25
Ala 15.0 14
Cys/2 9.6 10
Val 18.4 22
Met 1.1
Ile 11.1 13
Leu 17.0 18
Tyr 9.0 9
Phe 7.8 7
Lys 7.2 9
His 5.8 7
Arg 9.0 8
Trp 8.6 11
Total 2~2
*' See Japanese Patent Unexamined Publication No. 111688/1986.
-16-
2Q~L47~3
(D) Enzymatic ac~ivity
The measurements were made 8S follows: Elastin-decomposing
activity, by the m~thod of W. Ardelt et al. (Anal. Biochem 34, 1~0,
(1970); casein-decomposing activity, by the method of Non~to et al.
(Riken Report 34:6 381, (1958~); esterase-activity on BTEE (benzoyl-
tyrosine-ethyl ester) and TAME (tosyl-arginine-methyl ester), by the
method of B.C.W. Hammel (Can. J. Biochem. Physiol 37, 1393 (1959));
and activity on SAaPNA, by the method of J. Bieth et al. (Bivchem.
Med. 11, 350, (1974)). The results are shown in Table 2. For
comparison, activities of porcine elast~se n~de by Sigma, and bovine
chymotrypsin and bovine trypsin both made by Worthington are also
shown. As compared with porcine elastase, the human elastase obt~ined
in the present study was about the same in cRsein-decon~osing
activity, but considerRbly weaker in elastin-decomposing activity, the
value being about 1/10 - 1/20 of that of porcine elastase. As for
SA~PNA-decomposing activity, the human elsstase was weaker to some
extent, but it still showed strong activity. Such a tendency is
generally known of elastase-l. The human elRstase obtained in the
present study is presumed to be elastase-l not only from its having,
as mentioned above, ~he same amino acid composition as that of
elastase-l but also from its enzymatic 8c~ivity. Moreover, it did not
act on TAM~, a typical substrste for trypsin, and it showed only
slight activity on BTEE, a typical substrate or chymotrypsin.
However, its activity was about 1/5 of that of porcine elsstase ~ade
by Sigma. From these, no trypsin or chymotrypsin is considered to h~ve
been contained in said human elastase.
-17-
Z~4~3
W ~ ~ o o
~ _ ~
~ _, -'-c~
_
~ '
~ 'e ~ oo o
~ ~ ~_I ~ ~
~ a ~ +~
?
~
h * ~ U~ Lll
.~ ~ ~ ~ O ~ O
~ ~ _I ~ ~ O~ O
~0~ _
.~ ~ O
~*C~ ~ 'gOO ~
8 ~ _+, +, ~ j
~: . g.
C~-~
G)
~-
~td
~ .
~1 ,J h ~; ~ 31~
D _ .. ..
**
--1 8--
2~ 703
(3) Preparation of anti-El serum
In 0.25 ml of physiological saline was dissolved 0.25 mg of El,
which was then emulsified with 0.25 ml of Freund's complete adjuvant.
This was injected into a domestic rabbit (Japanese white) 5 times at
intervals of 3 weeks, and thereby antiserum was prepared. For
radioimmunoassay, a 65C00-fold dilution of said antiserum W8S used.
(4) Prepar~tion of '26I-labeled El
The '26I-labeling of El was performed by chloramine T method. To
5 ~ g of El ~ere added 1 mCi of Na'26I (made by Amersham) and 50 ~ 1
of 0.5 M phosphate buffer (pH 7.4). After these were mixed, 10 ~ 1 of
0.2% solution of chloramine T was added and the mixture was stirred
for 30 seconds at room temperature. Then, 50 ~ 1 of 0.25% solution of
sodium metabisulfite was added. After the solution was mixed, 10 ~ 1
each of 5Xo potassium iodide and 1% solution of bovine serum alb~mln
were added, which was then subjected to gel filtration [Sephadex G-50,
~ 0.9 x 25 cm, developing solution: 0.1 M phosphate buffer solution
(pH 7.~)], whereby '2sI-labeled El fraction ~as collected. To this
was added the measuring buffer solution mentioned later in (7) and
the mixture was allowed to stand overnight so as to bind Boc-TACK to
El. The solution was made into a 400000 dpm/ml preparation, ~hich was
then used for radioimmunoassay. The labeling rate was about 50~ and
the specific radioactivity was about 80 ~ Ci/~ g.
~5) Second antibody
Immunobead-goat anti-rabbit IgG (made by Bio-Rad) was made into
a 100 /Ig/ml preparation.
(6) ~el filtr~tion
To human serum was added '2ol-labeled El, which was incubated for
30 minutes at 37C. This was then subjected to gel filtration
[Ultrogel AcA 44 (made by IBF Biotechnichs)~ ~ 1.5 x 55cm, developing
solution: 0.1 M phosphate buffer (pH 7.1)]. The eluate was collected
-1 9-
~ 3l~7~3
in l-ml portions and the radioactivity in each fraction was measured.
(7) Radioimmunoassay
As for measuring buf~er solution, 0.01 M phosphate buffer (pH
7.4) containing 0.1 mM Boc-TACK, 0.5r/o sodium chloride, 0.05% sodium
azide and 0.2~o bovine serum albumin was llsed unless otherwise
mentioned. One hundred ~ 1 of st~ndard El solution (0.5 - 50ng/ml) or
sample serum was t~ken into a polystyrene tube. To this were added 100
~ 1 each of '26I-labeled El solution and anti-El serum, which was then
mixed and incubated for 3 hours at 37C. Next, 500 ~ 1 of the
preparation of the second antibody was added, which was mixed and
incubated for 30 minutes at 37C. ~he incubat~d product was
centrifuged (3000 rpm, 5 minutes), after which the supernat~nt ~as
removed by suction and the radioactivity of the precipitste was
measured.
II. Inhibition activity of Boc-TACK on the binding of serum inhibitor
to l2~I-labeled El
(1) Evaluation by gel filtration
When the human serum to which '2sI-labeled El not treated with
Boc-TACK had been added ~as subjected to gel filtr~tion, the l25l-
labeled El was all eluted in the fraction of its complex con~ining
a l-antitrypsin and a 2-macroglobulin (Fig. l-a); On the other hand,
most of the '2~ beled El trea~ed with Boc-TACK was elut~d in the
free fraction, and none of it was found in the complex fraction (Fig.
l-b).
(2) Evaluation by an immunological procedure (rsdioimmur~oassay)
Radioimmunoasssy W85 performed using a l-antitrypsin or a 2-
macroglobulin solution ia place o the standard El solution, whereby
the effect of these on the binding rate was examined. As for ~easuring
buffer solution, one mentioned in above-mentioned (7) was used in one
method (A m~thod), and one not con~aining Boc-TACK was used in another
--~0-
~ O~L~ O 3
method (B method), and one containing PMSF in place of Boc-TACK was
used in third method (C method) ~Fig. 2). In B method, the binding
rate decreased gradually with increases in serum inhibitor and similar
decreases were also seen in C method. On the other hand, in A method,
little change was observed in the binding rate and only a slight
decrease was seen with a 2-macroglobulin in a high concentr~tion.
III. Measurement of human serum El
(1) Standard curve and dilution curve of human serum
With regard to standard curve, as shown in Fig. 3, a good curve
of competitive reaction was observed within a range of 0.3 - 50 ng/ml,
and the sensitivity was 0.5 ng/ml. To evaluate the effect of
Boc-TACK on the immunological reaction of El, standard curves were
drawn of Boc-TACK-treated El and untreated El, and then comparison was
made between the two. As shown in Fig. 4, no difference was observed
bet~een the two, and even when Boc-TACK was bound to El, it did not
affect the immNnological reaction of El in any way.
Meanwhile, the dilution curve of hu~an serum was roughly in
parallel with the standard curve (Fig. 3).
(2) Speciiclty
Competitive reaction curves were drawn of serum inhibitor and El
complexes based on the results of radioimmNnoassay, and cross
reactivity of each complex was examined by making comparison with the
standard curve of El (Fig. 5). Cross reactivity of a l-antitrypsin,
a 2-macroglobulin and El complexes are shown in Table 3. When ~ross
reactivity of standard El is taken as 100%, the ~alues of El complex
containing a l-antitrypsin and tha~ containing a 2-macroglobulin are
26% and 2% or less, respectively. As shown in the resu~ts of
radioimmunoassay reported in [Atsuo Mura~a et al.: Japanese Journal
of Gastro-Enterology, 78, 1985 - 1993 (1981)3, cross resctivity was
observed only with El complex containing a l-antitrypsin. When acting
-21-
Z O~L~ )3
alone, neither a l-~ntitrypsin nor a 2-macroglobulin showed dny cross
reactivity.
Besides, as shown in Fig. 6, no cross reactivity was seen with
any of the animal sera.
T~ble 3
Cross reactivity of ~ ,-antitrypsin ( a ,-AT) . a 2-macroglobulin
( a 2-MG) and El complexes
_ __ _ _
Inhibitor or ElCross re~ctivity
complex wi~h it (%)
_ _ ._ _
El- a, -AT 26
al-AT (0.1
El-a 2-M& (2.0
a 2-MG (0.1
(3) Recovery of hum~n serum El
A specimen of human serum to which Boc-TACK-treated El had been
added beforehand was measured, ~nd thereby ~he recovery was
c~lculated. A good result WAS obtained~ with the recovery being 100.4
+ 8.9% as shown in Table 4.
-22
~ Y0 3
Table 4
Recovery of Boc-TACK-treated El added to human serum
Added amount Value ofRecovery
measurement
(ng/ml) (nB/ml)
0 2.9
0.5 3.6 10~
1.5 4.8 109
5.0 7.8 99
15.4 86
54.0 102
Average recovery 100.4+8.9 (%)
(4) Serum El in healthy humans
Serum El was measuret on 6 healthy adults. The average velue was
: 2.38 + 0.72 ng/ml, which was nearly in agreement wi~h the results
obtained wi~h a radioimmunoassay kit (made by Dain~bot) commercialIy
available. (Table 5)
~23-
Table 5
Serum El measured in healthy humans
.
Serum No. Serum El (ng/ml)
Method of With the kit
this invention
. _ _
1 3.~ ~.5
2 2.3 2.~
3 2.0 l.g
4 2.~
1.4 1.6
6 3.0 2.7
_ .
Average 2 38+0.72 2.08~0.46
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